A method for gene editing to regulate mosquito-borne virus infection in mosquitoes

Abstract

The present invention relates to a method for gene editing of mosquitoes to regulate mosquito-borne virus infection. Specifically, for the first time, the CRISPR / Cas9 technology is used to partially knockout the predicted MAL1 gene in Aedes aegypti, and Aedes aegypti with gene knockout is obtained. Subsequent functional verification experiments show that: compared with wild-type Aedes aegypti, the infection rate of the intestine of the gene-knockout Aedes aegypti after virus infection is significantly decreased, indicating that substances that inhibit the MAL1 gene can be used to prevent and control diseases transmitted by mosquitoes. The gene-knockout Aedes aegypti prepared by the present invention can be used as a research model for the interaction between mosquitoes and viruses and mosquito metabolism. At the same time, the success of gene editing of mosquitoes in the present invention also makes it possible to perform gene editing on more non-model organisms.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for gene editing to regulate mosquito-borne virus infection in mosquitoes. Background Art

[0002] In recent years, the global incidence of mosquito-borne infectious diseases such as dengue fever, Zika, and malaria has increased significantly, posing a major challenge to public health. At present, there are no effective treatment regimens and preventive vaccines for the vast majority of mosquito-borne infectious diseases during the internal incubation period (human infection period). The main prevention and control strategies are comprehensive prevention and control strategies mainly based on environmental control and chemical control during the external incubation period (mosquito infection period) to cut off the mosquito-borne transmission route. However, with the increasing resistance of mosquitoes to pesticides year by year and environmental protection issues, traditional prevention and control strategies have been challenged unprecedentedly. With the development of gene editing technology, the research on immune biological blockade of mosquitoes during the external incubation period has become an important research direction for blocking the transmission of mosquito-borne diseases today.

[0003] The CRISPR / Cas system is an acquired immune system of archaea. By cutting phage dsDNA with Cas proteins, the phage genome is destroyed to achieve the purpose of bacteria immune to phages. CRISPR / Cas9 is an endonuclease of the type II family of the Cas system, which can perform site-directed cleavage between the 3rd and 4th bases in the 5' direction of the genomic NGG PAM structure, causing double-strand breaks in DNA. Then, through the non-homologous end joining and homologous repair mechanisms of the cell DNA repair mechanism, the broken DNA strands are reconnected together, thereby realizing the editing of genomic DNA sequences. The emergence of CRISPR / Cas9 technology has provided a target-specific modification tool for molecular biological basic research in many aspects such as mosquito physiology, biochemistry, development, and the relationship between hosts and pathogens, bringing new opportunities for the development of mosquito control technologies.

[0004] In recent years, there have been some reports on the use of gene editing technologies to prevent the transmission of mosquito-borne diseases. For example, researchers from the U.S. Army's Institute of Collaborative Biotechnologies and the University of California, Santa Barbara used the CRISPR / Cas9 gene editing tool to target genes related to the fertility of male mosquitoes and conducted "sterilization" experiments on Aedes aegypti. They used a control method called the sterile insect technique to breed many sterile male mosquitoes and then released these insects. When female mosquitoes mate with sterile male mosquitoes, the viability of the former will be affected, thus reducing the size of the next generation. However, this prevention and control method requires continuous release of sterile male mosquitoes to suppress the mosquito population, which is restricted in practical applications. Moreover, similar "sterilization" tests have also been carried out in Brazil. It was found that after genetically edited male Aedes aegypti that theoretically can reduce the population of Aedes aegypti were released into the environment, some of the mosquito offspring still reached sexual maturity and hybridized with local mosquitoes, which also illustrates the instability and uncertainty of this method to a certain extent. Therefore, there is still a need to find a new method of using gene editing to prevent and control mosquito-borne infectious diseases. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for preventing and controlling mosquito-borne virus infections by gene editing for the extrinsic incubation period of mosquito-borne infectious diseases. By knocking out specific regions of the mosquito genome, the probability of mosquito-borne virus infection is reduced, providing a new direction for the treatment of such diseases.

[0006] The first object of the present invention is to provide the application of a substance that knocks out or partially knocks out the gene sequence shown in SEQ ID NO.2 or its homologous sequence in the preparation of products for the prevention and treatment of mosquito-borne infectious diseases.

[0007] Further, partial knockout includes knocking out the 73-99th bases of the gene sequence shown in SEQ ID NO.2 or its homologous sequence.

[0008] Further, the substance that knocks out or partially knocks out the gene sequence shown in SEQ ID NO.2 or its homologous sequence includes the CRISPR / Cas system, siRNA, shRNA, sequences encapsulated by lentivirus, etc.

[0009] Further, the CRISPR / Cas system includes sgRNA and Cas9 nuclease. The Cas9 nuclease uses the recognition and positioning of the sgRNA to complete the cleavage of the target gene fragment, resulting in the breakage of the target gene fragment in mosquitoes or mosquito cells, thereby undergoing homologous recombination repair or non-homologous end joining, and further causing base frameshift mutations to achieve the purpose of gene knockout.

[0010] Further, the sgRNA is designed with the sequence shown in SEQ ID NO.2 or a part of it as the target.

[0011] Furthermore, the sgRNA is designed against the target shown in SEQ ID NO.3.

[0012] Furthermore, the sequence of the sgRNA is as shown in SEQ ID NO.4.

[0013] Furthermore, the prevention and control of mosquito-borne infectious diseases include, but are not limited to, the step of editing mosquitoes or mosquito cells to reduce the probability of virus infection.

[0014] Furthermore, the mosquito-borne infectious diseases include, but are not limited to, dengue fever, Zika, malaria, filariasis, Japanese encephalitis, echinococcosis, ascariasis, yellow fever, chikungunya fever, etc., that is, the prevention and control of the infection of dengue virus (DENV), Zika virus (ZIKV), malaria parasite, filaria, Japanese encephalitis virus, Echinococcus granulosus, Ascaris, yellow fever virus, chikungunya virus (CHIKV), etc.

[0015] The present invention for the first time uses the CRISPR / Cas9 technology to knockout the MAL1 gene in Aedes aegypti, and obtains Aedes aegypti with partial knockout of the MAL1 gene. Subsequent functional verification experiments show that: compared with wild-type Aedes aegypti, the viral load of the Aedes aegypti with partial knockout of the MAL1 gene decreases after virus infection, indicating that substances that inhibit the MAL1 gene can be used to prevent and control mosquito-borne diseases. At the same time, the Aedes aegypti with partial knockout of the MAL1 gene prepared by the present invention can be used as an in vivo research model for the interaction between mosquitoes and viruses and the metabolism of mosquitoes. At the same time, the success of gene editing of mosquitoes in the present invention also makes it possible to perform gene editing on more non-model organisms.

[0016] The second object of the present invention is to provide a drug for preventing or treating mosquito-borne infectious diseases (for the mosquito infection period), containing a substance that knocks out or partially knocks out the gene sequence shown in SEQ ID NO.2 or its homologous sequence.

[0017] Furthermore, the drug for mosquito-borne infectious diseases is administered to mosquitoes or mosquito cells.

[0018] Furthermore, the substance that knocks out or partially knocks out the gene sequence shown in SEQ ID NO.2 or its homologous sequence includes the CRISPR / Cas system, siRNA, shRNA, lentivirus-packaged sequence, etc.

[0019] Furthermore, the CRISPR / Cas system includes sgRNA and Cas9 nuclease.

[0020] Furthermore, the sgRNA is designed against the sequence shown in SEQ ID NO.2 or a part of the sequence thereof as the target.

[0021] Further, the sgRNA is designed against the target shown in SEQ ID NO.3.

[0022] Further, the sequence of the sgRNA is as shown in SEQ ID NO.4.

[0023] Further, the mosquito-borne infectious disease drug is used for at least one of the following scenarios:

[0024] 1) Inhibiting virus infection in mosquitoes or mosquito cells;

[0025] 2) Preventing and treating diseases transmitted by mosquitoes;

[0026] 3) Preparing transgenic mosquitoes with a reduced level of virus infection.

[0027] The third object of the present invention is to provide a method for reducing the level of virus infection in mosquitoes or mosquito cells, including the step of knocking out or partially knocking out (the full length or a fragment) the gene sequence shown in SEQ ID NO.2 or its homologous sequence in mosquitoes or mosquito cells.

[0028] Further, it includes the step of knocking out the AATGAAGATCTTTGTTCCACTTCTAAG fragment in SEQ ID NO.2 or its homologous sequence in mosquitoes or mosquito cells.

[0029] Further, the knocking-out method includes, but is not limited to, replacing the sequence containing SEQ ID NO.5 with the sequence containing SEQ ID NO.6, targeting and knocking out the AATGAAGATCTTTGTTCCACTTCTAAG fragment, etc.

[0030] Of course, the present invention also provides a method for preparing transgenic mosquitoes with a reduced level of virus infection, including 1) or 2) as follows:

[0031] 1) Inhibiting the activity of MAL1 protein in mosquitoes or reducing the content of MAL1 protein in mosquitoes;

[0032] 2) Replacing the DNA molecule corresponding to SEQ ID NO.5 in the sequence listing in mosquitoes with the DNA molecule corresponding to SEQ ID NO.6 in the sequence listing.

[0033] Further, the method further includes the step of microinjecting the sgRNA and Cas9 nuclease into mosquito eggs. Preferably, the final concentration of the sgRNA is 20 - 60 ng / ul, and the final concentration of the Cas9 nuclease is 100 - 500 ng / ul.

[0034] Further, the method further includes the step of using the gene-edited mosquitoes as parents and hybridizing them with wild-type or gene-edited mosquitoes.

[0035] In the present invention, the above-mentioned mosquitoes include, but are not limited to, Aedes aegypti.

[0036] The fourth object of the present invention is to provide a method for constructing a mosquito model or a mosquito cell model with reduced viral infection level, including the step of knocking out or partially knocking out the gene sequence shown in SEQ ID NO.2 or its homologous sequence on the genome of mosquitoes or mosquito cells.

[0037] Furthermore, after the knocking-out step, it further includes the steps of incubating mosquito cells, and then hybridizing the obtained individuals. The above-mentioned steps of incubation and hybridization can be repeated at least once until a knockout homozygote is obtained, that is, the mosquito model is obtained.

[0038] Furthermore, the specific construction method of the mosquito model includes:

[0039] S1. Treat mosquito eggs with the CRISPR system containing the sgRNA shown in SEQ ID NO.4 to obtain gene-edited mosquito eggs;

[0040] S2. Incubate the mosquito eggs to obtain parental mosquitoes, and hybridize the parental mosquitoes with wild-type mosquitoes to obtain offspring mosquitoes;

[0041] S3. Repeat step S2, and hybridize the obtained offspring with the offspring until a knockout homozygote (a homozygote with the 73rd to 99th bases in SEQ ID NO.2 knocked out) is obtained to obtain the mosquito model.

[0042] The fifth object of the present invention is to provide the mosquito model or mosquito cell model obtained by the above construction method.

[0043] The sixth object of the present invention is to provide the application of the above mosquito model or mosquito cell model in studying mosquito metabolism or the interaction between mosquitoes and viruses.

[0044] By means of the above solution, the present invention has at least the following advantages:

[0045] 1. The present invention discovers for the first time that MAL1 can regulate the infection of mosquito-borne viruses. By knocking out or partially knocking out the MAL1 gene in mosquitoes or mosquito cells, the viral load in mosquitoes or mosquito cells can be reduced, thereby preventing and controlling diseases transmitted by mosquitoes.

[0046] 2. The present invention for the first time uses the CRISPR / Cas9 technology to construct Aedes aegypti with partial knockout of the MAL1 gene. The obtained gene knockout strain is helpful for the study of genes, proteins or other functions in mosquitoes, can be used as an individual model of mosquitoes for mosquito metabolism research, can also provide an individual model of mosquitoes for the study of mosquito resistance to pathogenic microorganism infections, and can also provide an individual model of mosquitoes for the study of the interaction between genes regulated by the MAL1 gene and pathogenic microorganisms and mosquito vector hosts.

[0047] 3. The CRISPR / Cas9 gene knockout technology adopted by the present invention is more effective than technical means such as gene silencing and interference. Due to many advantages such as strong operability, low cost, and wide application range, this technology is one of the most promising gene therapy technologies in clinical and application.

[0048] 4. The present invention can precisely and efficiently partially knockout the MAL1 gene by using gRNA primers.

[0049] 5. The method of microinjection of mosquito eggs adopted by the present invention is more suitable for gene editing of insects. A relatively large number of gene knockout mosquito strains can be obtained by one injection.

[0050] 6. The method of screening genomic DNA from pupal cuticles adopted by the present invention does no harm to mosquito individuals and can quickly realize the screening of knockout mosquitoes. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in conjunction with the drawings.

[0052] Figure 1 It is a schematic diagram of mosquito egg injection.

[0053] Figure 2 It is a flow chart for screening gene knockout mosquitoes.

[0054] Figure 3 It is the sequencing peak map of knockout mosquitoes and the comparison with the sequence of wild-type mosquitoes. Among them, a is the boundary peak map of single and double peaks in the sequencing of FO generation chimeras; b is the partial sequence alignment map of molecular cloning sequencing of FO generation chimeras; c is the partial sequence alignment map of molecular cloning sequencing of F1 generation heterozygotes; d is the schematic diagram of sequence alignment between MAL1 gene knockout mosquitoes and wild mosquitoes.

[0055] Figure 4 It is a flow chart of virus infection of MAL1 gene knockout mosquitoes and wild-type mosquitoes.

[0056] Figure 5 It is the change of virus infection level in knockout mosquitoes and wild-type mosquitoes after infection with dengue virus. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0057] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited are not intended to limit the present invention.

[0058] The sequence information related to the present invention is as follows:

[0059] AAEL009524, alpha-amylase, MAL1 (SEQ ID NO.1)

[0060] MKIFVPLLSFLLAGLTTGLDWWEHGNFYQVYPRSFKDSDGDGIGDLDGVTEKLKYLKDIGMDGVWLSPIFSSPMADFGYDISNFREIQTEYGDLDAFQRLSDKCKQLGLHLILDFVPNHTSDQHEYFKKSVQKDETYKDFYVWHPGVHGPNNTKVPPSNWISVFRGSSWEWNEERQEFYLHQFLKEQPDLNYRNPAVVEEMKNVLRYWLDRGVSGFRIDAVPYLFESDIIDGRYRNEPESRTTDDPENPAYLVHTQTMDQPETYDMIYQWRAVLDEYSKTDNRTRIMMTEGYTSLPKIIEFFGNATANGAQIPFNFEVISNVKKNSTGADFATYVKRWLDAKPANRRSNWVLGNHDNNRLGSRLGENKIDLYNIALQTLPDIAVTYYGEEIGMLDQWIPWNETVDPAACRSDEASYSAYSRDPARTPMQWDSGKNAGFSKAAKTWLPVADNYKTLNVKIQDRARKSHLKIFKKLTKYRKRQILTEGDIDIKVSGENLLVYKRKVDKVGYVVVALNFGTEPVALGLSSLFDRADQRMQVVVSSNRVSTPDNVWVDVDNYVLIGESGIVLQYLWGKNPIVS

[0061] LOC5572111, probable maltase [Aedes aegypti (yellow fever mosquito)], MAL1 (SEQ ID NO.2)

[0062] TTAGCGAACATGGTCATCATTCAACTGACATCGGTTGCTCTAGGCATAGAGTGCACTGATCGGAACAGGCAGA ATG

[0063] Target sequence recognized by sgRNA (SEQ ID NO.3)

[0064] GCTAGGAGGAAGCTTAGAAG

[0065] Nucleotide sequence of sgRNA (SEQ ID NO.4)

[0066] TAATACGACTCACTATAGGCTAGGAGGAAGCTTAGAAGGTTTTAGA GCTAGAAATAGC

[0067] Partial verified sequence of wild-type Aedes aegypti MAL1 gene (SEQ ID NO.5)

[0068] TTAGCGAACATGGTCATCATTCAACTGACATCGGTTGCTCTAGGCATAGAGTGCACTGATCGGAACAGGCAGA ATG AAGATCTTTGTTCCACTTCTAAGCTTCCTCCTAGCAGGACTAACCACCGGGTTGGACTGGTGGGAACATGGAAACTTCTACCAAGTTTACCCAAGATCCTTCAAGGACTCCGACGGCGACGGTATCGGGGATCTGGACGGTAAGATCAGGATCTAAGCTTACAATTTATCATTAAGAATTATTTCTTTCTCTAGGAGTCACCGAAAAGCTGAAATATCTGAAAGACATCGGCATGGACGGAGTTTGGTTGTCACCGATTTTCTCTTCTCCGATGGCTGATTTTGGCT

[0069] Partial verified sequence of knockout Aedes aegypti MAL1 gene (SEQ ID NO.6)

[0070] TTAGCGAACATGGTCATCATTCAACTGACATCGGTTGCTCTAGGCATAGAGTGCACTGATCGGAACAGGCAGCTTCCTCCTAGCAGGACTAACCACCGGGTTGGACTGGTGGGAACATGGAAACTTCTACCAAGTTTACCCAAGATCCTTCAAGGACTCCGACGGCGACGGTATCGGGGATCTGGACGGTAAGATCAGGATCTAAGCTTACAATTTATCATTAAGAATTATTTCTTTCTCTAGGAGTCACCGAAAAGCTGAAATATCTGAAAGACATCGGCATGGACGGAGTTTGGTTGTCACCGATTTTCTCTTCTCCGATGGCTGATTTTGGCT

[0071] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0072] The Aedes aegypti in the following examples was collected in Haikou City, Hainan Province and propagated in the breeding room.

[0073] Example 1 Construction method of Aedes aegypti with MAL1 gene knockout

[0074] I. Preparation of sgRNA

[0075] 1. Design of sgRNA target

[0076] According to the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), the possible DNA sequence of the Aedes aegypti MAL1 gene (Gene ID: 5572111, amino acid sequence is shown in SEQ ID NO.1, nucleotide sequence is shown in SEQ ID NO.2) was obtained, and the sgRNA guiding sequence was designed based on this DNA sequence. The target sequence of sgRNA is as follows:

[0077] sgRNA target sequence: 5’-GCTAGGAGGAAGCTTAGAAG-3’ (SEQ ID NO.3);

[0078] 2. Synthesis of sgRNA sequence

[0079] Artificially synthesize the sgRNA sequence:

[0080] sgRNA sequence (SEQ ID NO.4):

[0081] TAATACGACTCACTATAGGCTAGGAGGAAGCTTAGAAGGTTTTAGA GCTAGAAATAGC。

[0082] II. Preparation of Aedes aegypti with MAL1 Gene Knockout

[0083] 1. Preparation before Microinjection

[0084] 1) Preparation of RNP Complex

[0085] RNP complex: Mix TrueCut TM Cas9 Protein v2 (10 μg / μL) (Invitrogen, catalog number 01005198) with the sgRNA solution (solvent is water) to obtain a mixture with a final concentration of 40 ng / μL for sgRNA and a final concentration of 300 ng / μL for Cas9 nuclease.

[0086] Mix the above mixture and incubate at 37 °C for 15 minutes to obtain the ribonucleoprotein (RNP) complex.

[0087] 2) Preparation of Mosquito Eggs to be Injected

[0088] Collect the incompletely developed Aedes aegypti mosquito eggs within 30 minutes after they are laid. Use a No. 0 suture needle to arrange the mosquito eggs neatly, with the end where the gonad is located (the small head end) on the same side.

[0089] Use transparent double-sided tape to transfer and fix the mosquito eggs on a 20×20 cm glass slide. Dehydrate for 30 - 60 s. When the mosquito eggs are observed to be slightly sunken, add Halocarbon oil 27 (SIGMA, catalog number H8773) to maintain the humidity of the mosquito eggs.

[0090] 3) Preparation of Microinjection Needle

[0091] Use a Microloader TM pipette tip (Eppendorf, catalog number 5242956003) to add the RNP complex in 1) to the Femtotip II microinjection needle (Eppendorf, catalog number 5242957000). Fix the microinjection needle on a BV-10 needle grinder (SUTTER, catalog number BV10-2300) and gently grind a small opening at the tip of the needle.

[0092] Install the microinjection needle after opening on the needle holder of the FemtoJet 4i air pump (Eppendorf, product number 5252KN102540). Fix the needle holder on the robotic arm (Eppendorf, product number 5193KN302559) at an appropriate angle (usually 20 - 30 degrees), and control the microinjection needle using the TransferMan4r joystick (Eppendorf, product number 5193KN302559).

[0093] 2. Microinjection of mosquito eggs

[0094] Place the mosquito eggs prepared in step 2) under a stereomicroscope and adjust to an appropriate magnification, usually 100 - 200 times. Use the joystick to control the microinjection needle and inject the RNP complex into the end where the gonad of the mosquito egg is located, with the mosquito egg slightly bulging but not bursting. A total of 2244 mosquito eggs were injected, which are the G0 generation. The schematic diagram of mosquito egg injection is as Figure 1 shown. After wetting the injected mosquito eggs with dechlorinated water, gently pick the mosquito eggs off the double-sided tape with a fine brush and transfer them to a wet thick filter paper for incubation.

[0095] III. Screening and sequencing verification of Aedes aegypti with MAL1 gene knockout

[0096] 1. Rearing of F0 generation mosquitoes

[0097] Place the injected mosquito eggs in the rearing room (relative humidity 75 ± 5%; temperature 28 ± 1°C; day-night time ratio: 14h:10h) for 5 days of rest and then incubate. Fill a deep enamel tray with dechlorinated water as deep as possible, add a little feed, immerse the thick filter paper with eggs in the tray, and cover for rearing after complete hatching. Scrape the film once in the morning and once in the evening to keep the feed sufficient. A total of 36 mosquitoes (19♀, 17♂, F0 generation) hatched, with a survival rate of 1.60%. After pupation, place the pupae in mosquito rearing tubes respectively, feed them with cotton balls soaked in 8% sugar water, and after eclosion, take the pupal skins shed and number them correspondingly for subsequent detection.

[0098] 2. Design primers for MAL1 gene amplification

[0099] Design a pair of PCR amplification primers for the MAL1 gene according to the DNA sequence of the Aedes aegypti MAL1 gene above. The primer sequences are as follows:

[0100] F: 5’-TTCGCGCCACATCGTTATTC-3’;

[0101] R: 5’-AGCCAAAATCAGCCATCGGA-3’.

[0102] 3. PCR amplification of the wild-type Aedes aegypti MAL1 gene

[0103] Extract the genomic DNA of wild - type Aedes aegypti (Takara, catalog number 9765), and perform PCR amplification using primers F and R designed in step 2 to obtain the PCR product. The PCR reaction system (Takara, catalog number RR901A) is shown in Table 1, and the PCR reaction conditions are shown in Table 2.

[0104] Table 1 PCR reaction system

[0105] Component Dosage Premix Taq 12.5 μl F 0.5 μl R 0.5 μl DNA template 2 μl dd H2O 9.5 μl Total 25 μl

[0106] Table 2 PCR reaction conditions

[0107]

[0108] Sequence the PCR product with clear and bright bands to obtain a partial sequence of the wild - type Aedes aegypti gene. The product length is 419 bp, and positions 57 - 419 of the sequence, a total of 363 bp, are part of the MAL1 gene (SEQ ID NO.5).

[0109] 4. Screening and verification of homozygous MAL1 gene - knockout Aedes aegypti

[0110] Extract the genomic DNA of the pupal cuticle of the F0 - generation knockout Aedes aegypti in step three - 1. Since the amount of pupal cuticle DNA is small, design a pair of nested PCR primers based on the MAL1 gene PCR amplification primers FR. The sequences of the nested PCR primers are as follows:

[0111] NestF: 5’ - TCCATTGATTAGCGAACATGGT - 3’;

[0112] NestR: 5’ - AGTCCTTGAAGGATCTTGGGT - 3’.

[0113] Detect the mutation of the MAL1 gene sequence in the pupal cuticle of the knockout Aedes aegypti by nested PCR amplification. The primers, reaction system, and conditions for the first - round PCR amplification are the same as those in steps 2 and 3 of step three. The nested PCR amplification primers are NestFR. Dilute the first - round PCR product 100 - fold and use it as the amplification template. The reaction system is the same as that in step 3 of step three, and the reaction conditions are shown in Table 3.

[0114] Table 3 PCR reaction conditions

[0115]

[0116] Sequence the nested PCR products. Since the F0 generation knockout mosquitoes are chimeras, the sequencing results cannot be directly compared with the MAL1 gene sequence of wild-type Aedes aegypti. First, observe whether there is a single-double peak boundary at the sgRNA target knockout site in the sequencing peak diagram. The sequence before the sgRNA target is normally sequenced and the peak diagram is a single peak. If the knockout is successful, the sequence after the sgRNA target is disordered and the peak diagram is an irregular double peak, so as to judge whether the mosquito corresponding to the pupal skin is a MAL1 gene knockout mosquito. The screening process of gene knockout mosquitoes is shown in Figure 2 .

[0117] The results showed that: A total of 8 mosquitoes may have been successfully knocked out, namely ♀9, ♀10, ♀13, ♀14, ♀15, ♀16, ♀18, ♂17, and the gene editing rate was 22.22%. The single-double peak boundary of the sequencing peak diagram is shown in Figure 3 a in. Mate them with wild-type Aedes aegypti respectively, feed them with blood 3 days later, and collect eggs 3 days later. They can be fed with blood and eggs collected multiple times (G1 generation). At the same time, select the PCR product of the pupal skin DNA of one mosquito ♀15 (F0 generation, chimera) for gel cutting and recovery (Tiangen Biotech Co., Ltd., product number DP209), connect the recovered product to the vector (TransGen Biotech Co., Ltd., product number CT101), select bacterial monoclonal colonies for sequencing after spreading on the plate, and further verify whether it is knocked out after sequence alignment. Since the F0 generation is a chimera, the sequencing results of 30 clones of the pupal skin DNA of ♀15 showed that some of the monoclonal sequences had base deletions, two genotypes, with deletions of 8 bp and 11 bp respectively. The sequence alignment is shown in Figure 3 b in.

[0118] Incubate the G1 generation eggs produced by ♀15 (F0 generation) again according to the method in step 3(1). Screen the Aedes aegypti with gene knockout in the F1 generation according to the above nested PCR-sequencing peak diagram-molecular cloning-sequence alignment method, and select the pupal skin DNA of ♀1 (F1 generation, heterozygote) for molecular cloning. Since the F1 generation is a heterozygote, the sequencing results of 30 clones showed that some of the monoclonal sequences had base deletions, with a deletion of 27 bp. The sequence alignment is shown in Figure 3 c in. Mate ♀1 (F1 generation) with male wild-type Aedes aegypti and collect eggs by feeding with blood multiple times (G2 generation).

[0119] Incubate the G2 generation eggs again according to the method in step 3(1). Use the pupal skin (F2 generation, heterozygote) to verify by sequencing again according to the above method. Select the female Aedes aegypti with base deletions and mate them with male Aedes aegypti, and collect eggs by feeding with blood multiple times (G3 generation).

[0120] Incubate G3 eggs again according to the method in step 3(1), use the pupal skins (F3 generation, coexistence of heterozygotes and homozygotes) to verify by sequencing again according to the above method. Select homozygous female and male Aedes aegypti of F3 generation with base deletion (all monoclonal sequencing sequences have base deletion) according to the sequencing results of molecular cloning for mating, and collect eggs after multiple blood feedings (G4 generation).

[0121] Incubate G4 eggs according to the method in step 3(1) to obtain a homozygous Aedes aegypti strain with MAL1 gene knockout (F4 generation), with a 27bp sequence deletion. A schematic diagram of the sequence of some knockout strain Aedes aegypti is shown in Figure 3 d in. Collect eggs after multiple blood feedings, expand the breeding, and prepare for subsequent infection verification experiments.

[0122] The difference between the genomic sequences of the knockout mosquito strain and the wild mosquito strain is only that there is a 27bp fragment deletion in the MAL1 gene sequence (SEQ ID NO.5) (located in the exon, including the promoter). This deletion fragment is located at positions 73-99 of SEQ ID NO.5. The mutated MAL1 gene sequence in the knockout mosquito strain is as shown in SEQ ID NO.6 in the sequence listing.

[0123] Hereinafter, the MAL1 knockout strain Aedes aegypti is simply referred to as the KO strain, and the wild strain Aedes aegypti is simply referred to as the WT strain.

[0124] Example 2 Detection of virus level after Aedes aegypti with MAL1 gene knockout is infected with virus

[0125] I. Mosquito virus blood meal infection experiment

[0126] Test mosquito strains: WT strain Aedes aegypti, KO strain Aedes aegypti.

[0127] Experimental method: Conduct an oral virus blood meal infection experiment using mosquitoes that have emerged for 3-5 days. After infection, the mosquitoes are fed with cotton balls soaked in 5% sugar water. The mosquito virus infection process is shown in Figure 4 .

[0128] Specific steps:

[0129] 1. Cut off the supply of sugar water 24 hours before the infection experiment to promote the mosquitoes to suck blood quickly in a hungry state;

[0130] 2. Transfer the mosquitoes that have had their sugar water supply cut off to the infection laboratory;

[0131] 3. Add 200ul fetal bovine serum to the blood meal cup, then add 4ml fresh sterile defibrinated sheep blood, and then add an equal volume of dengue virus suspension and mix well;

[0132] 4. Gently unfold the sealing film to simulate the skin to seal the mouth of the blood meal cup. Connect the blood meal cup to a peristaltic pump (one end is connected to a 37°C water bath) and place it in the infected mosquito cage;

[0133] After 2 hours of blood supply, the mosquitoes were sucked out with a mosquito suction device, anesthetized with carbon dioxide, and placed on ice. Well-fed female mosquitoes were selected and raised separately.

[0134] II. Detection of virus level after virus infection

[0135] About 30 mosquitoes each were dissected at 3 days, 6 days, 9 days, and 12 days after infection to obtain midgut samples. After extracting RNA, virus detection was carried out. The detection kit used was a one-step fluorescence quantitative detection kit (probe method, TransGen Biotech, product number AQ211).

[0136] The dengue virus primers and probes are as follows (recorded in the literature "Study on the co-infection of dengue virus type 1 and dengue virus type 2 in C6 / 36 cells and Aedes albopictus"):

[0137] D2-F: 5’-AATTAGAGAGCAGATCTCTGATGAA-3’;

[0138] D2-R: 5’-AGCATTCCAAGTGAGAATCTCTTTGT-3’;

[0139] D2-P: HEX-GCTGTTGCACAGTTGACACGCG-BHQ1.

[0140] Table 4 Reaction system

[0141] Component Dosage 2×PerfectStart*Probe One-Step qPCR SuperMix 10 μl TransScripr*Probe One-Step RT / RI Enzyme Mix 0.4 μl F(10 μM) 0.4 μl R(10 μM) 0.4 μl Probe(1 μM) 1 μl RNA template 2 μl RNase-free Water 5.4 μl Total 20 μl

[0142] Table 5 Reaction conditions

[0143]

[0144] The results showed that after infection with dengue virus, the infection rate of the midgut of Aedes aegypti KO strain was lower than that of the WT strain, and the difference in the infection rates between the two was statistically significant (P<0.05). The specific differences are as Figure 5 shown.

[0145] Obviously, the above embodiments are only examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Application of the sgRNA shown in SEQ ID NO.4 in the preparation of a product for the prevention and treatment of mosquito-borne infectious diseases; the mosquito-borne infectious disease is dengue virus infecting Aedes aegypti; the mosquito in the mosquito-borne infectious disease is Aedes aegypti.

2. A drug for mosquito-borne infectious diseases, characterized in that: The mosquito-borne infectious disease drugs contain the following: sgRNA shown in SEQ ID NO.

4.

3. The mosquito-borne infectious disease drug according to claim 2, characterized in that: The mosquito-borne infectious disease drug is administered to mosquitoes or mosquito cells.

4. A method for constructing a mosquito model or a mosquito cell model with reduced viral infection level, characterized in that: The following steps are involved: S1. Using a CRISPR system containing the sgRNA shown in SEQ ID NO.4 to treat mosquito eggs to obtain gene-edited mosquito eggs; S2. Hatching the gene-edited mosquito eggs to obtain parent mosquitoes, hybridizing the parent mosquitoes with wild-type mosquitoes, repeating the process at least once, and obtaining offspring mosquitoes; S3, hybridizing the progeny mosquitoes until a knockout homozygote is obtained to obtain the mosquito model; The mosquito-borne infectious disease is dengue virus infecting Aedes aegypti; the mosquito in the mosquito-borne infectious disease is Aedes aegypti.

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